DNA double strand breaks (DSBs) are the most dangerous forms of DNA damage, as they can form chromosomal translocations or deletions if improperly repaired, or cause cell death if they persist unrepaired. DNA DSB repair pathways involve coordination of a large cohort of proteins, many of which are essential for these pathways. However, many DNA repair proteins can be inactivated without causing significant DSB repair defects. We reason that in many cases this is due to the presence of compensatory pathways. One of the most dramatic examples is revealed by the combined deficiency in the histone variant H2AX and the non-homologous end joining (NHEJ) protein XLF. H2AX recruits many DNA damage response proteins to DSB sites, but deficiency in H2AX does not cause an overt defect in NHEJ. Similarly, deficiency in XLF results in relatively minor defects in NHEJ. However, combined deficiency of H2AX and XLF results in a severe defect in NHEJ-mediated DSB repair. These findings suggest that H2AX and XLF function have compensatory or synergistic functions during DNA DSB repair. We reason that compounds that inhibit XLF or proteins in the same pathway as XLF would inhibit NHEJ specifically in H2AX-deficient cells. Here, we will use a novel cell line based approach for assaying DNA DSB repair to develop a high throughput screen for compounds that can inhibit NHEJ. In collaboration with the NIH Chemical Genomics Center, we will use this screen to identify chemicals that inhibit NHEJ specifically in H2AX-deficient cells. We will also establish approaches for identifying the targets of these compounds. Completion of these studies will be important in several ways. First, our findings will establish that agents that do not affect DSB repair in normal cells may affect DSB repair and genomic stability in rare cells that have lesions that lead to reduced H2AX expression. Second, as many tumors inactivate H2AX, compounds identified through our screen could be used therapeutically to sensitize these tumor cells to genotoxic agents. Finally, in addition to H2AX, there are other DNA repair proteins whose inactivation does not lead to severe DSB repair defects. Our approach could be used to both establish the existence and identify the components of compensatory pathways for these proteins, and to identify compounds that inhibit these pathways.